R EGULA R ARTICLES

Expression of the "Skin-Type" Desmosomal Cadherin DSCl Is Closely Linked to the Keratinization of Epithelial Tissues During Mouse Development

Ian A. King, Toby J. O 'Brien, and Roger S. Buxton Division of Eukaryotic Molecular Gellctics. National Illstitute for Mcdical R.esearch, London, U.K.

DesITlosomal junctions contain two classes of desmo­SOD1al cadherin, the desmocollins and the desnlO­gleins, each of which occurs as three distinct iso­forD1s . To investigate the role of the "skin-type" desDlosomal cadherins (desmocollin 1 and desmo­glein 1) in the formation of keratinized epithelial structures, we have now cloned full-length mouse desDlocollin 1 complementary deoxyribonucleic acid and examined the expression of desmocollin 1 and desDloglein 1 and messages during murine embryonic development by ill situ hybridization. In the general body epidermis, desmocollin 1 and desmoglein 1 transcripts both showed considerable upregulation at 15.5 d, which is after the onset of stratification and before the start of keratinization. Before this the epidermis expressed low levels of desmocollin 1 mes­sage , although the desmoglein 1 signal was always stronger and more extensive. In the tongue, expres-

Recent mol ecular clon ing of the maj or g lycoprotcin constitu ents of dcsmosomcs (desmocollins o r DSCs, and d eslll ogic ins or DSGs) fi'om bovin c (Goo dwin el ai, 1990; Koch el ai, '1990; Collin s el ai, '199 1; Koch CI (/1, 1991 a, 1991 b; M cchanic el ai,

1991; Koch ct a i, 1992; Legan cl ai , 1994; Yuc el al 1995), human (Amagai et ai, 199 1;, Nil les el ai, 1991; Pa rke r el ai, 1991; Wheeler ef aI , 1991; King el ai, 1993a; Theis el al; 1993 ; Kawa mura el ai, 1994; King, 1994; Schiifer el ai, 1994; King el ai , 1995) , and murine sources (B uxton el ai, 1994b; Ish.ikawa et ai, 1994; Lorim er el ai, 1994) has shown that bo th compon cnts a rc m embcrs of thc cadherin family of ccll adhesion molccul es (Magcc and Buxton , 1991; Buxton '1I1d Magee, 1992; Buxton cl ai, 1993; Koch and Franke, 1994). Extracellul ad y, the desmosomal cadh erin s comprisc four cadh erin-likc repcats and an cxtrace llular ancho r domain, and they contain ac idi c Ca2 + -binding sitcs. The cytoplasmic domains have distinctive stru c tural fcatures that impart fun ctional specificity

Manuscript rcccived March 25, '1996; revised J Llnc 14, 1996; accepted for publication Junc 26, 1996,

Reprint requests to: Ian A. Ki ng, Division of Eukaryotic Molecular Genetics, National Insti tute for Medical R.cscarch. T he Ridgeway. Mill HiU, London NW7 1AA. U.K.

Abbreviations: DSC. dcsmocollin ; DSG, desmoglcin ; E. crnbryonic age ill days ; RT, reve rse trn nscriptasc.

sion of desmocollin 1 luessage occurred several days after desmoglein 1 and coincided with the formation of the keratinizing filiform papillae. Desmoglein 1 message was also detected in epithelial tissues in which desmocollin 1 was absent, suggesting that expression of the two "skin-type" desmosomal cad­herins was not tightly coupled during embryonic development. HUluan desmocollin 1 monoclonal an­tibodies that cross-reacted with mouse skin and tongue indicated that desluocollin 1 protein was first expressed in those outermost epithelial cells destined to form the keratinized layers of the stratmu cor­neum or the papillae. The results suggest that expres­sion of desmocollin 1 is closely associated with the keratinization of epithelial tissues during mouse de­velopluent. Key Ivol·ds: desmosomeldeslltocolli"ldesllto­glein. ] 111"est Del'lllatol107:531-538, 1996

to the desmosomal cadherins as compared to the classica l cadherins (T royanovsky e/ ai, 1993, 1994a, 1994b) .

It is now c1eal' that the DSCs and the DSGs each occur as three genetica ll y distinct subtypcs (Buxton CI ai, 1994; Koch and Franke . 1994) and that a ll six desm osom al cadhe rin genes are closely linked on chrom osom e 18 in thc human (Amemann el ai, 1991, 1992a, 1992 b; Overhauser el ai, 1993; Wang e/ ai, 1994; Am agai el a11995; Simrak el ai , 1995) and th c mouse (Buxton el ai, 1994b; Ishikawa et ai, 1994) . Moreover, the expression of particul ar desm osomaI cadh crin gen es appears to corre late to some extent w ith thc mode of epi thelial differentiation (Arnemann el ai, 1993; Legan elal, 1994; Schafcr el ai, 1994; King ct ai, 1995; Nubcr el ai, 1995). DSC2 and DSG2 were shown to bc expressed in severa l epithelial tissucs. myocardium , and some cpithclial cell lines by reverse transcl;ptase­polylllemse chain rejlction (RT-PCR) ,1IlaJysis of bovine tissues (Legan CI ai, 1994), and by RNase protection assays on both bovin e and human tissues and ccll lines (Schafer e/ ai, 1994; Nubcr et al 1995).

The other desmosoma l cadhcrins appea l' to be expresscd , in addition to DSC2 and DSG2, in particul ar subsets of cpithclial tissues, alth ough th ere are somc discrepancies in the reported expression patte rns . DSC3 was detectcd in bovine epidermis , esophagus, tonguc, trachea, and rumen by R T-PCR (Legan e/ ai, 1994) and in human epidermis, tonsil, esophagus, sa livary g land, and som e epith e lial carci no m as by RN ase protection analys is (Nuber el al. 1995) , We o bservcd hum an DSC3 in the living layers

0022-202X/96/ S10.S0 • Copyright 1996 by T hc Society fo r Investigative Dermatology. Inc.

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532 KJNG ET AL

of foreskin e pide rmis by ill sitll hybridization and also in buccal

mucosa, esoph agus, and cervix by immunofluorescen ce with spe­c ific anti-peptide antibodies (King ef aI, 1995) . DSG3, the a u toan­

tigen in p e mph.ig us v ulgaris (Amagai et ai , 1991), was d etected in a similar subset of tissues to DSC3 b y RNase protection assays on human and bovin e mate rial (Schafer e/ aI, 1994), su ggesting th at

expression of thi s desmosomal cadh e rin pair mig h t b e assoc iated

w ith epith e li al stratification. T he "skin -typ e" desmosomal cadhe rins (DSC1 and DSG1) h ave

the most restricted distribu tion among the m emb e rs of th.is gene fa miJ y. DSC1 mRNA was o nl y fo und in bovine e pidermis and

tongue b y northe rn blotting, a lt h ough DSC1 transcripts were detected in som e other tiss u es by RT-PCR (Legan et aI, 1994). DSCI was on ly d etected in huma n epidermis and lymph node by RN ase protection (Nuber et aI, 1995), and o u r immunofluorescent study on human stratified e pithe lia suggested th at Dscl was re ­stricted to th e keratiniz ing epiderm is of th e skin (King et ai, 1993 b, 1995). DSG1 , the autoantigen in pe mphig u s foliaceo us (Stanley et aI, 1986), was observed in bovin e and human e pide rmis, tongu e, and esoph agus b y RNase protection analysi s (Schafer et aI, 1994), implyi n g a slig htly less restricted distribution than DSC1. T h e "skin-type" desmosomal cadhcrins also exhi b it character istic ex­pression p atterns in strati fied e pithe lia b ecau se both gen es ar e expressed in the n o nprolife rative suprabasal compartme n t (Parrish et ti l, 1986; Jones et ti l, 1987; JGng et ti l 1991 ; Arnem ann et til, 1993; King et ti l, 1993b; T h e is et ti l, 1993). In bovine n ose epide rmis DSCl expression sta r ted in t h e first suprabasal layer (Legan et ti l, 1994), whereas in h uman foreskin epide rmis it was n ot d etected

until th e upper spinous/granular layers (King e/ aI, 1991, 1993b, 1995). Because of this discrepancy, it was not clear w h eth er

expression of DSCl was associated w ith the stratification of epith e lial tissues or with th e keratini zation of stratified e pithe lia .

To address this question , we have n ow iso lated cDNA clones for m o u se DSCl and h ave examined its expression during e mbryonic deve lopm e nt by ill sitll hybridization and by immunofluorescence . To d etermine wheth e r expression of th e two "skin- type" desmo­somal cadh erins is c lose ly coupled during e pithelial morphogenes is ,

we h ave a lso examine d th e developmental express ion of DSG1 .

MATEIUALS AND M ETHODS

RT-PCR Tota l RNA was isolatcd (Chomczynski and Saachi, 1987) fi:om poolcd ncwborn mo usc skin (2 g), and po lyA + RNA was isolatcd on o ligo (dT)-ceUulosc using a RNA microprcp kit (Pharmacia, Piscataway, NJ) . First strand eDNA synthesis w ith randonl h eXal11CrS and pert were as used before to isolate human DSC3 cDNA (King ef ai, 1995) cxccpt that the PC R. pril11 CrS were lIsed at lower conccntrations to irnprove specificity.

1](52 (5 ' -CTTGGAAAlGA/GTGGGCNATC/TCTTGC- 3') and IK53 (5'-CAGAGTGTGTCCTCTAATGGATTC-3') we re used at fina l concen­trations of 0.69 J.LM and 0.06 J.LM, respectively. R. T-PCR. products were ge l-purified by electroe lu tion , subcloned into pBluescript as described prev iously (IG ng ef ai , 1995), and sequenced (Sangcr <I aI, 1977) after alka li denaturation using Seq uenase 2.0 (Amersham , Arlington H eights , IL).

Isolation of eDNA Clones AUni-Zap XR cDNA library from 11-wk­o ld fema le mouse skin (Stratagenc, La J o ll a, CA) was screened by fi lter hybrid ization with the mouse DSCl RT-PC R product after excision with BalllH I and Hilldlll and ge l purification . Probe labc ling. fi ltcr hybridi zation, and washing were as described previously (King cf ai, 1995). Six positive cloncs (Tl to T6) idcntified in thc first ro und of screening were plaquc­purifI ed, and pBluescript phagemids were cxc ised using the ExAssist/SOLR systcm (Stratagcnc) . cDNA inserts were sized by rcstriction ana lysis and charactcrized by PCR.. For the lattcr, we used primcrs obtained from thc origina l RT-PCR c10nc seq ucnce and lei-om thc pBluescrip t multiple cloning site to identi fY those c10ncs containing the longest 5' cD NA sequence (T3) and the lo ngest 3' -scqucnce (Tl). TOB2 (5' -GGAGTGCTAGGAGCAGC-3') and IKl18 (5'-ACGACTCACTATAGGGCG-3') were used to identi fY 5 ' clones and TOB4 (5'-GCA TGTAAACAAAATGCACAG-3'), and \K117 (5'CAAAAGCTGGAGCTCCAC-3') were used to identifY 3' clones. C loncs Tl and T3 were sequenced on both strands, and scqucnccs wcre analyzed using the software of the Genetics Computer Group (program man ual for the GCG package, versio n 7. Madison, W I) .

I .. Sit .. Hybridization T he age of mouse cmbryos (Pa rkcs strain) was estim ated from the appeara ncc of the vaginal plug. Embryos and excised

THE JOURNAL OF INVESTIGATIVE DER.MATOLOGY

tissues were fixed in 4°/', parafonnaldehyde for 18 h , washed o nce (30 min) in sa line and tw icc in sa line/ethanol (1:1 ), and transferred to 70% ethanol before embedding in paraffin wax. Sagittal and parasagittal sections of whole cmbryos and scctions fi·om excised tiss ues were pretrcated as described prev iously (Arnemann ef aI, 1993).

DSC l riboprobes wcre synthesized from a pBluescript subclo ne contain ­ing the 5' BalllHI fi·agmcnt (1.5 kb) of clone T3 . DSG l riboprobes were derived fi:om a 681 base pairs (bp) EcolU clone (Buxton ef ai , 1994b) containin g 3' sequence. C lones were linearizcd with appropriate enzytlleS, and antisense and sense 3sS_labelcd riboprobes we re generated using T7 (Amersham) or T3 (l3oehringer, Indianapo li s, IN) RNA polymerase and a commercial RNA- labeling kit (Amersham). Probes were reduced in size by alka li trea tment at 60°C for 30 min (King ef aI, 1995) and were purified by gcl fIl trati on and ethano l precipitation. Scctions were hyb ridized with riboprobes (1-2 X 106 dpm per J.LI) at 60°C for 18 h and washed as previo usly described (A rnemann ef ai , 1993; King ef ai , 1995) to a maximum of 65 °C, and slides were exposcd to X- Omat AR x-ray fi lm for 18 h. Slides were then coatcd with LM-l emulsion (Amersham) and exposed for up to 1 wk before being developed and counterstained w ith 0.02% toluidine blue.

Western Blotting afFusion Proteins The expression constructs con­taining extraceUular domains of human DSC1, DSC2, and DSC3 in pGEX-2TK (Pharmacia) wcre describcd previously (King ef ai, 1995). Single colonies of recombinant Escherichia w li werc grown in 2 ml of TY medium with 2% glucose and 100 (.Lg ampici llin pe r mi. and GST fusion proteins were induced with 0.1 mM isopropyl-J3- D-thiogalactopyranoside for 2 h . l3acteria were co llected by centri fugation and were lysed by boiling the pell e t in sodium dodecyl sulfate sample buffer. Lysatcs wcre fractionared on 8'Yo sodium dodecyl sulf.1te-po lyacrylamide gels and transferred to ni troce llulose. Fil ters we re blocked with 1 'x. bovine serull1 albumin (BSA) in phosphatc-buffered sa line (PBS) and incubated with hybridoma supem atant (1 :1 0 to 1:100 in 1% BSA/PBS) and thell with horseradish pcroxidase­conjugated goat anti-mouse IgG (l3io-R.ad , Richmond, CA) diluted 1:1000 in 1% BSA/PBS. Bound antibodies were dctected by chemiluminescence (Enhanccd C hemiluminescence systcm , Amersha m) and exposurc of the filtcrs to x-ray fi lm .

Indirect Immunofluorescence Embryos and excised tissues were mounted in T issuc-Tek OCT embedding compound (M iles. Elkhart, IN), snap-frozen in isopentane/liquid N 2 , and sto red at - 70°C . Cryostat sections (5 J.Lm) were air-dried, blockcd with 10% no rmal rabb it se rum in PBS , and incuba ted with monocion,d antibodi es (hybridoma supcrnatant) di luted 1:4 with 1% BSA/PBS conta ining 0.1')1" eth ylcnediaminc tetraacetic acid. Eth ylcl1cdiarnine tctrnacetic flcid was previo usly shown to e nhance stain.ing w ith thcsc DSCl monoclonals (King cf al. 1993 b). Scctions wcre then incubated with fluorcscein isothiocyanate- conjugated rabbit anti-mouse IgG (Dakopatts, Copcnhagen , Denmark) dilutcd 1 :40 in 1% I3SA/ PBS, washed, and mounted in Vcctashicld (Vector Laboratories, Peterborough, U.K.) .

RESULTS

Cloning and Sequence of Mouse DSC1 cDNA T h e original clone was generate d by R T -PCR on polyA + RNA &om n ewborn m ouse skin u sing t h e re dundant sense o li gon ucleotide prime r IK52 and th e antise n se primer IK5 3, d es igned to amplify the C-term inal domain of the sh o rter DSC splice variants (King et ti l, 1995). This generated a single PCR prod u c t of 423 bp, w hic h was ide ntified as th e probable mouse ortholog of DSC1 after subcloning and se­que ncing. It had 84'Yu nucleotide identity w ith human DSC1 (King et til 1993a; IGng, 1994). T his clon e was used to screen a A. mouse cDNA libra ry and isolate two overlapping clones, T1 (3203 bp) and T3 (2941 bp), w hich were sequenced o n b oth strands and used to

d educe the comple te sequence of murine DSC1 (Fig 1) . T3 was found to contain an open read ing fi·ame with in-frame stop codon of2661 bp and was ide ntified as the lo n ger sp lice variant ("a" forn1) of DSC1. I t coded for a n unprocessed polypeptide of 886 amino acids, w hic h , afte r proteolysis at the pre dicted cleavage site, would

yie ld a mature DSC of752 residues. C lone Tl overlapped with the T3 seque n ce from position 859 to 2920 apart from an additional 46-bp alternatively spliced sequence with premature stop cod on that produces the sh orter variant ("b" form) of DSC1 , which is predic ted to con tain 698 amino ac ids (Fig 1) . T1 contained an additio n al 1096 bp of 3' -un translated sequence, includin g a termi­nal polyA sequence (not shown) , but it did n ot sh are the stretch of 2 1 A's found at the end of clone T3. It is n ot clear w heth e r thi reflects multiple polyadenylation sites, beca use the on ly consensus

VOL. 107, NO.4 OCTOI3ER 1996

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Figure 1. Nucleotide and predicted amino acid sequence of mu­rine DSC1. The sequence was deduced from overlapping cDNA clones T3 and Tl as described in the text. T he start of the clone T I and the original RT-PCR product are shown. Potential N-glycosylation sites are indicated by e. The predicted N renninus of the processed polypeptide is indicated with L:::,. and the putative cell adhesion recogni tion sequencc by O . T he ttan sm e mbrane sequence is Itefl/lily IIl1derlilled and the position of the alternative ly spliced ex on (boxell) is indicated by ! . This sequence has been given the accession number X97986 in the EMBL/ Genbank database.

polyadenylation sequence in T3 occurred around the TAA stop codon, whereas Tl had an AAATAA motif 1075 bp further downstream.

The predicted amino acid sequence of mouse Dsc1a showed 82% and 80% overall amino acid identity with the human and bovine proteins, respectively. Figure 2 compares the three sequences dire ctly using th e Pil e-up program of GCG (DeveJ·e ux et ai, 1984). T h e major structural motifs are conserved across all three species. In particular, the putative cell adhesion recognition site (Y AT) is unchanged, supporting our contention that this may be the most reliable indicator ofDsc subtype (Buxton el ai, 1994a). Two of the four potential N-glycosylation sites in m ouse Dsc1 arc conserved. Inte restingly, mouse Dsc1 lacks one of the two partially conserved amino acid repeats present in the cytoplasmic domain of human and bovine Dsc1 (Fig 2) . The significance of this difference is not yet cl ear, a lthough bovine but not human Dsc2 was found to contain three repeats of the equivalent sequence (Legan el ai, 1994).

DSC1 EXPRESS ION IN DEVELOPM ENT 533

Nevertheless, this leads to slightly shorter Dsc1 proteins (752 and 698 residues) in the mouse compared to man and cow, which contain 760/706 and 7611707 amino acids, respectively.

Expression of DSCl Message During Mouse Development To determine whether the expression of DSCI was linked to epithelia l stratification and/ or keratinization during mouse devel­opment, sections of whole 11.5-d to 16.5-d (Ell.5 to E16.5) embryos and excised skin and tongue fi·om older animal s were examined by ill silll hybridization using an anti sense 35S-labeled riboprobe. DSGl was examin ed at the same time to determine whether similar regulatory processes governed the developmental expression of the two "skin-type" desmosomal cadh erins.

Visual inspection of autoradiographs prepared from hybridi zed sections indicated that DSC1 sign al was first detected at 13.5 d as a single spot in the region of the external nare (Fig 3). It was not detected in the gen eral body epidermis l1Jltil 15.5 d, when DSC1 showed considerable upregulation and the signal fornled a clear o utlin e around the embryo sections . DSC1 signal was entirely restricted to the epidermis and was not detected in internal embryonic structures even by 16.5 d. The DSG1 signa l followed a generally similar pattern except that it was also detected in the oral cavity at E15.5 (Fig 3).

Higher resolution of the hybridization signals was obtained by microscopic examination after coatin g the sectio ns with autoradio­graphic emulsion. Stratification of the genera l body epiderm is started at about 13.5 d and was more pronounced in the ventral surf.Kes of the embryo. At this stage, the epithelium close to the developing external nares was a lready multilayered and expressed DSCI and, to a greater extent, DSG1 messages (Fig 4(/,[,) . Although both genes were supra basa lly expressed, DSG1 tran­scripts were present deeper in th.is nasal epithe lium than those of DSC1. A low level of DSGI message was also detected over the thicker part of the lip epidermis (Fig 4d), but DSC1 was not detected in these areas (Fig 4c). By 14.5 d the vcntra l epidermis was clearly stratifying, and a low level of DSC1 labeling was now observed in some areas (Fig 4g), but the epidermal DSGl signal (Fig 4") was stronger and more extensive than that of DSC1 at th is stage. The specialized follicles of the tacti le vibrissae above the eye and on the upper lip (Fig 4e,j) were strongly labeled by both riboprobes, reflecting the early development of these whiskers , whereas the sparse body hair buds were sti ll unlabeled.

DSCI message levels were markedly upregulatcd at 15.5 d, and transcripts were generally present in the outer cell layers of the stratifying epidermis (Fig 41). Comparison of adjacent sections indicated that DSCI expression con sistently started higher in the epidermis than did DSGI (Fig 4i,)). In the upper lip , DSC1 transcripts were strongly expressed in vibrissa follicles that had been sectioned through or just above the hair bulb (Fig 4k). Follicles sectioned nearer the skin slll·face showed progressively less DSC1 signal, and this was concentrated over the inn er cell layers of the follicle. DSG1 m essage was expJ·essed in a similar pattern (Fig 41) although it was m ore prominent in deeper sectio ned fo llicles and was less restricted to the iuner cell layers of more superficially sectioned foUicles . Longitudinal sections through vibrissa follicles in 16.5-d excised upper lip skin (Fig 4/11) showed that DSCl message was not present in the proliferating cells of the hair matrL,. [c was prominent in the outer cell la yers of the upper hair bulb and continued into the keratinizing cells of the inner root sheath . DSC1 message was not detected in the cells of the ou ter root sheath. DSGI message was expressed in a similar manner (Fig 4,,), although it consisten tly started deeper in the hair bulb and was more prominent in the outer layer of the inner root sheath.

In 16.5-d terminal phalanges, DSCl m essage was not detected in the proliferating cell layers of the nail matrix (Fig 40) but was restricted to those sllpertlcial ce ll layers over the prm";mal half of the nail bed starting within the nail fold. DSGI message was also found in these areas (Fig 4p) although. as in the hair follicle, express ion ofDSGl started deeper in the epithelium and transcripts

S34 JaNG ET AL

Figure 2. Pile-up comparison of mouse, human, and bovil1e Dscl amino acid sequences. Identica l resi­dues are indicated with an • and con­served cysteine residues arc s"nded. The two partia lly conserved cytoplasmic re­peats in human and bovine Dsc1 (see text) are ollcdi"cd wil" s" nded boxes , and the alterna tively spliced exon is in brack­cis . • indica te conserved N-glycosylation sites. Other symbols as in Fig 1.

rrosc1a hdsch bds ch

mdsc h hdscl. bdsch

mdsc1 a hdsc1 a bdsch

T H E JOURNA L OF INVESTIGATI V E DEI1.MATOLOGY

I 100 MAVACMPGST FSKQLLF FLLVLVL FCOACQKVSLHVPSHLKAETPVGKVNLEECLKSPSL I LSSDPAFR I LEDGT I YTTHOLLLSSEKRGFS I LL5OGO MALASAAPGS I FCKQLL FSLLVL TLLCoACQKVYLRVPSHLCAETLVGKVNLEEtLKSASL I RS50PAFR I LEDGS I YTTHDL I LSSERKSFS I FLSDGC MAVASAAPGS I FQKCLLFSLLVL I LFCoACcK I SLCVPSHLRAEALVGKVNLKECLCSASL I LSSDPDFR I LEDGS I YTTHDLVLSSG. KSFS I LL50SQ •• '* ••••• '* •• *** **** '* ••• *** ***** •• *. * *** ***. ****** ••••••••

101 • _ 200 GQECKKLEWLSAREKKVFRKRHTKEPVHNRSKRR~AP I PCsLMENSLGPFPCH I QC I CSDAACNYT I frS I SGPGODKEPYNLFYI EKDTGO I YCTRS I RREQQE I KWLSARENKSPKKRHTKOT ALKRSKRR~AP I PASLHENSLGPFPQHVCC I cSOAAcNYT I FYS I SGPGVOKEPFNLFYI EKOTGO I FCrRS I

GQG~KE I E II :E!GGK~P . ~~~M!OAVLR~T~~~~!~!~~:~;~~~~~~~~~V~~V~!~!!~~!!!:!~!~~~~~~;~F~:: F !~~!~! F~!~!! 201 [JIJ 300 ORECYOCFLVYGY A TT AOGYAPOYPLPLL FKVEOONONAPYFETKL TVFSVPEN'CRSGTSVGOVT AIOKOEPGTLHTRLKYK I LQQI POcPKHFS I HPDT DREKYECFAL YGYA TT AOGYAPEYPLPL II K I EDONDNAPYFEHRVT I FTVPEN~RSGTSVGKVT A TOLDEPOTLHTRLKYK I LCCI PDHPKHFS I HPOT

ORECYCE:P 1 ! A !~!!!~~!!~E!:;:; VF~;~~~~~::!~~NKL !V ~T~:;~r.T~!!~~K~!: I ~l~~~!;~!~:~!~!~~!:NNPR H FTVHPO T

301 400 mds c , . GV I TTTTPLLDREKCOTYKL VHEVROHGGCPFGL FTTGT I T I SLEOENDNSPYFTCTSYTTEVEENR lOVE I LRHWHDCOLPNTPHSKAVYT I LKGNEN hdsc l. GVI TTTTPFLOREKcilTYCL I HEVROHGGOPFGLFNTGT I T I SLEOENDNPPSFTETSYVTEVEENR lOVE I LRHKVCOCOLPNTPHSKAVYK I LCGWEN IxIsc 1. GVI TTTTPLLDREKCoTYKL I HEVROHGGQPFGLFNTGT I T I SLEOENONAPYFTETSYTVEVEENR lOVE I LRMAVHDHDLPNTPHSRAVYC I LCGNEN

•••••••••••••.•••• '* * ••• ** •• *.**** ***.* •••• ***** '* .* ••• * •••• * •••••• *. '* ... ******** *.* *. ****

401 500 md SC 18 CiolFI( I TTDPHTNEGIIL'¢wKPLNYEVSRQVTLQ I GVLNEAQFTNAANAQ. PPTH~TTVTVIC I KDROEGPEC:QPPVI(V I QSKOGLPAGQELlGYKAVDPE hdsc l . GN F I I STOPNTNEGVLtwKPLNYEVNRCVI LCVGV I NEAQFSKAASSC. TPTHCTTTVTVK II D50EGPECHPPVKV I CSCOGFPAGOELLGYKALOPE

~sc, 8 ~T:I(! S!~~~!~;A ~:~~~~~!;~N:~~:~ I ~~L~;~~.~ A~ VNSKT TT !~f!!~!~~K~ H~;~:;.~::y!~!~!E~~L~T~T;::~!!! ~~;

SOl _ 600 mdscl. TSSGEGLRYEHVGOEON~FE I NK I TGOLRTVKVLORESKFVKNNCYN I SWA TOTAGRS\:TGTl WLLEDFNDHPPQ I • OKEVT ICaCEKD FAVLEP IDL hdsc I 6 I SSGEGLRYCKLGOEDN~FE I NCHTGDLRTLKVLDRESKFVKNNQYN I SWAVOAVGRSQTGT L WHLOOYNDHAPC I . OKEVT I CaNNED FAVLKPVOP

bdsc 1 a RG T ~:~:~!KK I ~:~~~~:!~E y !~~; K!V~~;~~:!T :~~~~~!~V~~ I ~F~AO ~:~g!~!;~F; eOKNO HPpa I KQE! l !!SR HOKDYWLEPTOQ

rrdsc1e hdsc18 bdsc h

rrdsch hdsch bdsch

rrdsc1 e hdsch bdsc1a

~~~DNGPPFCFLLONSSSKL~TLESCDGKRAI LRCRHNLNYNHSVP I CI CORHGFSAKHVLSVRV:¢lcTTPTEtRMAVKEERDAK. PN II LG_ OGPENGPPFCF FLDNSASKN~N I EEKDGKT A I LRCRCNLOYNHSVP I CI KDRHGLVATHHL TVRvj;oasTPSE~HKDKSTRDVR. PNVI LGR~AI LAM OGPONGPPFCF I LDNSASKL~TVETRDGKT AI LRGRCOLOYOYYTVPI CI KORHGASA TH I LPVRVtotT I PSE¢RHPSKLSREAALANVFLGK~A I LAM ••• ••••••• •••• ..... '* ••• ...... .. '* •• * ••••• *.. .. ....... ii • • ' .. ...... *.. .. •• * •••••

. _ mmilllmgmfu~[rn VLGSALLL,~1 L FT~F~VTTTKRTVKKCFPDOVAQCNL I VSNTEGPGEEVTEAN I RLPTCT AN I alTSHSVGTLGGQG I KTQQSFEHVKGGHTLESHKGG. VLGSVLLL~ I L FT~F¢V. T AKRTVKK¢FPED I AcCNL I VSNTEGPGEEVTEAN I RLPHcTSN I !;oTSMSVGTVGGCG I KTQQSFEHVKGGYTLDSNKGGG

~;~~V;;;~!;~!::'f~ . !V~K!~~~f::E~V :~~~:!~!~!;~:~;;~:~!~~: T~!S~V~!! I !~~! L~~~~V~!~!::~~~~~Y~~OAH ~~~G

i:::;::i!;!!!mimi~! !!:!!i"m,;!ii!!!IIli!

•••••••• VLGAAEPGRYA YTOIICTFIQPRLGE HQT LE SVKGVGCGOT GRY A YTDIICS F TCPR L GE HCTLESVKGV •• TOTGRYTYSO~HNFTCPRLGE

••••• * ....... * ...

ESI RGHTL I KN KVYL~GCDEEHKHfEDYV~YNYEGKGSLAGS ~s I RGHTLI K~ KVYLCGCAEEHKHCEDYVRPYNYEGKGSMAGS

ES I RGHTLVKN KVYLCGCDEEHKLtEOYVRSYSYEGKGSVAGS ***.* •• *... .***~'*. **** .**** * ****** ****.

909 RQEEEGLEFLOCLEPKFRTLAKT~ RCEEEGLE F LO H LEPKfR TLAKT~ I KK

, RCEEEGLOFLDHLEPKFRTLAKTJ:VKK *.******. * •• ***.****.**." *-

were detected in less differentiated cell layers than w ere those of DSC1 .

DSC1 DSG1

In the oral cavity DSCl transcripts were not detected at 13.5 d (Fig Sa), although a low level of DSGl m essage was already present in the more distal oral epithelium, particularly the invag i­natin g lip furrow band (Fig Sb) . At 15.5 d, when epidermal DSCl m essage leve ls were upregul ated, DSC1 transcripts were observed in the epithe ljum over the upper and lower lips (Fig Se), but expression ceased at their inner boundari es and did not extend significantly into the oral epithelium . DSC1 m essage was not yet expressed in th e tongue. In contrast, DSG1 transcripts w ere strongly expressed throughout the o ral epithelium and in the m ore superficial layers on the do rsal surt:1ce of the tongue (Fig Sj). Formation of the keratiniz ing fi liform papillae started at 17 .5 d, and DSCI transcripts were now detected in the lingual epithelium for the first time (Fig 6a). At this stage, DSC1 m essage was restric ted to cells near the outer surface of the developing papillae, wh ereas DSG1 m essage was m ore wide ly distributed in the epith elium (Fig 6b) . As the papillae differentiated and the surf.1ce layers keratinjzed, DSCl m essage levels in creased and assumed a less superfi cial location (Fig 6e,g).

Exprcssion of Dscl Protcin During Mousc DcvcloplTlcnt T he four m ono clonal antibodies (LHsO, A4, B13, and G1 7) and the anti-peptide (IKP1) antibody previo usly generated against human Dsc1 (King e( aI, 1993b, 1995) were tested aga inst m o use skin by indirect immunofluo rescen ce, and two cross-reacting antibodies (A4 and B13) were identified. The isoform specificity of these reagents was ch ecked by weste rn blotting against glutathione-S­transferase fusion proteins containing the entire extracellular do­m ains of the three human Dscs (Fig 7) . Both antibodies were specific for D sc1 and did not recognize Dsc2 or Dsc3 fusion

E13.5

E14 .5

E15.5

E16.5

f----I 1cm

Figure 3. Autoradiographs of mouse embryo (E13.S to E16.5) sagittal sections hybridized with DSCl or DSGl antisense ribo­probes. After hybridiza tion and washing, slides were exposed to X-Omat AR x-ray film for 18 h. Note tha t DSC1 message is markedly upregulated at E15 .5 and is confined to the epidermis whereas DSG1 message is also strongly expressed in the oral cavity at this stage. Prior to this, DSCl and DSG1 sig nals arc restricted to a "spot" over the external nare.

VOL. 107. NO. 4 0 TOl3ER 1996 DSC I EXPR.ESS ION IN DEVELOPMENT 535

DSC 1 DSG 1 DSC 1 DSG 1

E13·5

E14·5

E15·5

E16·5

Figure 4. Photomicrographs of embryonic mouse epidermis hybridized with DSCl or DSGl antisense riboprobes. After hybridization and washing, slides were dipped in LM-l ell1ulsion and exposed for up to 10 d before developing and countersta ining with toluidine blue. Adjacent sagitta l or parasagi ttal sections through the developing ex terna l narc (a, b). muzzle (c, d) , upper lip (eJ) . ventral epidermis and foo t (g, ") . ventral epidermis and geni tal tubercle (iJ), upper lip (k, l) , and longitudinal sec tions through vibrissa follicles in the upper lip (11/ , 1/) and the developing nail in terminal phalanges ({) ,fl) arc sbown. VE, ventral epidermis; VF, vi brissa fo llicle; F. foo t; G. genital tubercle; I. Inner root shee th: 0 , outer root sheath: N, nail bed: DP. dermal papilla. Scale bar, 100 /km.

proteins, and these were thereforc used to cxa mine the expression of Dsc1 pro tein duJ"ing mouse development.

In the superficial body epide rmis, Dscl protein was not detected until E15.5. Staining was res tricted to the o uterm ost layc r of th e stra tified epithe lium , leaving several lower layers of cell s unsta ined (Fig 8a) . At this stage, the Dsc1-positive cells showed mainly cell surface staining , but they displaycd no overall o rganization. By 16.5 d , Dsc1 stainin g was stronger (Fig 8/1). Several of the o uter cell layers expressed Dsc1 pro te in , and thesc Dsc1-positive cells had nov.r started to fl atten and to o rganize into rcgular arrays of stacked cells precedin g the ir tc rminal difFerentiation into keratinized stra­tUn) corneum cells . O rganiza tion of the oute r epide rmal cell layers into a stratum corneum w as m ore advanced at 17.5 d (Fig 8e) , and the epiderm al staining pattern closcly resembled that in thc new­born mouse.

Dsc1 protein was detected in th e tongue at a later stage of development than in the skin , but a comparable developmental process was involved . Dsc1 protein was o bserved first at E17.5, several days after thc stratifi ca ti on of the lingual epithe lium. Initially, staining was detected in the outermost ceils, w hich were starting to form the keratinizing papillae (Fig 8t1) . By 19.5 d , the papillae were well differentiated and Dsc1 sta inin g was stronger, being presen t in severa l cell layers that closely followed the outline of the papillary surf.,ce of the tongue. T he Dsc1-positivc cell s, hov.rever, were still restricted to the upper layers of th e lin gual epithelium (Fig 8e). These results indicate that expression of Dsc1

prote in III the skin and ton gue is restri cted to those cells that are destined to form the keratinjzed layers of the stratum com eum and the filiform papillae.

DIscussrON Only one mouse DSC cDNA sequence has been reported previ­ous ly (Buxton el ai, 1994b; Lorimer CI ai, 1994). and this was shown to en code thc ortholog of the human and bovine Dscs (Dsc2) thar. are widely expressed in epithe lial tissu es, tumors, and cell lines (Nuber el ai, 1995). In this report, we have charactcJ"ized a sccond m ouse Dsc, which has only 53% amin o acid identi ty with m ouse Dsc2 and is clearly the murine equivalen t of human and bovine Dscl . Apart fi'o m the high (80 - 82%) overall amino acid identities, the three type 1 Dscs share the sam e putative ceU adhesion recognition sequen ce (Y AT) that distinguishes Dsc1 from the other Dsc isoform s (FAT in Dsc2 and YAS in Dsc3). T he conservation of this sequence supports thc v iew that it m ay be of fi.mctional significance in conferring adhesive specifi city o n the individual Dsc subtypes (King cl ai, 1995; Y ue el ai, 1995). M ouse Dsc1 exhibi ts the sam e two splice forms found in all the other Dscs so fa r cloned and sequenced, confinning the evolu tionary importance of the altemative splicing event. Its fun c tional relevance remains unclear, however, because the shorter splice variant ("b" form) has so f., r been fo und to be ineffec tive in plakogIobin binding, ancho rage of intermediate fil am ents, or plaque fo rmation (Troyanovsky CI ai, 1993 , 1994a, 1994b) .

536 K ING ET AI-

DSC 1 DSG 1

a T

b

6~ ~

.. ) ... .1

'" ~

E13·5

U U c d

T T .' -. f! '-....v' U , LJ

E14·5

e

E15·5

9

E16·5

F ig urc 5. Photomicrographs of cmbryonic mouse oral cavity hy­bridizcd with DSCl and DSGI anti-scnsc riboprobcs. T h e m ore di sta l po rtio n of the ora l cavity is shown. N o te the strong DSGl signal in tile oral epithelium, w hereas the DSC 1 signal ceases at the in ner boundaries of tllC lips . • ,. the lip furrow band. T, tongue; U , upper lip. Senl" lin ,., '100 fl.Jl )'

We have examined the expression of DSCI message by ill Sitll

hybridization on sagittal and parasagittal sec tions of whole m o use embryos, because this permits semiquantitative comparison of m essage levels in a wide range of ti ssues in the sa m e hybridization expel;men t. T his approach was feasib le only up to E 16 .5 because of th e size and onset of oss ification in o lder embryos. T he fi rst major findin g from these experiments was the considerable upregulation of DSC1 in the general body epidermis at 15.5 d . At thi s stage of development, DSC1 expression was enti re ly restricted to the epidermis. T hu s, general epidermal ex press io n of DSC1 transcripts occurred after the onset of epidermal stratification at £ 13.5 and preceded the onset of epidermal keratinization at about 1.7.5 d. Before 15 .5 d , a very low level ofDSCl m essage was detectable in the more stratified parts of the muzzle and limb epidermis, although higher levels were detected in the already multil ayered epid ermis over the external n ares. T he ta ctile vibrissae emerge at about E17 .5, at least 10 d before the pelage hairs, and this accounts for the precocio us expression of DSC l message in th e whisker follicl es .

T he on ly other ti ss ue in w hich DSCl message was detected was the tongue. Transcripts here were not detected un til E17.5, wh ich is well afte r the stratifi cation of the lingual epith e lium and co in­cided with the onse t of ke ra tinization and the form ation of fi li fo rm papilla e. The delay be tween expression ofDSClmessage and onset of keratiniza tion in the skin was not o bserved in the tongue, and this may reflec t the different m orphogenetic pathways in these epithelia .

T he immuno fluorescent loca lization of Dsc'l during epithe li al morphogenesis confirmed th e close associati on of Dsc1 expression with the fo rmation of keratini zed structures . In both skin and

THE JOU RNAL OF INVESTIGATIV E DEI1..MATOLOGY

DSC 1 DSG 1

a b

E17·5

C d

E19·5

e f

NB

Adult

F ig urc 6. Photomicrographs of tonguc hybridizcd with DSCl and DSGl anti-scnse r iboprobcs . Longitud in al sections fi'om EI 7," (a,b), E1Y.S (r,d) , and newborn to ngue (NI3) ("J) and transverse sections frol11 ad ul t to ngue (g,lI) :Ire shown. No te the appeara nce of DSC 1 mcssage in superfic ial ce ll s of E 17.5 to ngue co inciding w ith the fo rlll ation of the filifo rm pa pill ac . Senle bar, '100 (J.m.

tongue, Dsc1 was first detected in the outerm ost cells of au epith elium tha t was already stratifi ed bm had yet to keratinize. Dsc1 stainin g in creased with the formation of the keratinizing omer layers, indicating that ex pression of Dscl may be a m o re re liable marker than express ion of Dsgl and may even be a necessary prerequisite for this parti cul ar kind of epithelial difFerentiatio n.

T he second majo r findin g to em erge from the curre nt work was th at ex press io n of DSC 1 and DSG1 m essages in the developing mou se embryo was no t as tightly coupled as might have been anti cipated £i'om recen t reports (Schafer ci nl. 1994; Nuber cl nl, 1995) . T he latter stud ies exa mined a ran ge of ad ul t human and bovine tissues, tumors, and cell lin cs that contain desm osom es and confirmed that at least one DSC and o ne DSG isoform, in addirion to the plaque compon en ts, were req uircd to fOITn fu nctional desm osomes . M oreover. th ose ce ll types express ing o ne o r two add itiona l DSC iso fo rms also expressed the equi va lent, :ldditional DSGs. T hu s, particular DSC and DSG isoforms appea red to be constitutively expressed in pairs. O ur data indicate, however, that ex press ion of the two "skin- type" desm osomal cad herins (DSCl and DSG1) does no t occur simu lta neously. We have consistently observed ex press ion of DSGI message before DSC1 , both tempo­rall y in partic ular ti ssues and spatia II )' du rin g epithe li al m o rphogen­esis. T he most striking example of the former was rh e tongue, in

VOL. 107. NO.4 O CTOBER 19%

200 -

116-97 -

66-

45 -

A9 913 '------', ,-' ----~

--

2 3 4 5 6

Figure 7. Western blotting of glutathione-S-transferase fusion proteins containing the entire extracellular domains of the three human Dsc isoforms. Lysates containing fusion pro teins o f Dsc l (lnll es I , 4) . D sc2 (Inll es 2, 5), and Dsc3 (Inll es 3, 6) were trans fe rred to ni trocellulose and e ither stained w ith amido black (AB) or probed with the m onoclonal an ti b o dy B1 3 and dctected with the Enhanced C hemiluminescence system (5- 5 exposure) . T he antibody spec ifi cally recognizes the Dscl fusion protein (~) a nd some degradation products but docs no t recogni ze Dsc2 o r D sc3 .

w hic h high levels o f DSG1 message w ere present at 15.5 d but DSCl was not de tec ted until E17.5. DSGl message was also expressed in some epithelial sites in which DSC 1 was n o t present, su c h as the oral cavi ty (Fig 5), the epithelium between the fu sed eyelids, the interdig it;11 epithelium, the o lfactory epithelium, and th e g astric epithe lium (IA King, unpublished observations). In embryonic epithe li a expressing both DSG1 and DSC 1 messages , DSGl was usually expressed at a deeper level in the epithelium than D SC 1. This was m ost apparent in the nasal epithelium close to the ext e rnal nares. It appears th en that within epithe lial ti ssues there are cells e xpressing DSGl without concomitant expression ofDSC1 . I/I situ h ybridization studies on adult mouse skin ["om diffe rent body sites (IA King, unpublished observati ons) and adult tongue (Fig 6) have provided similar results, indicating that express ion of DS G1 message before D SCl is no t peculiar to embryonic tissues .

T h e expression ofDSG l before DSCl may not be specific for the " skin-type" desmosomal cadherins, and it wiLl be interes ting to de te rnline whe ther the other DSG I DSC isoform pairs have a similar relationship. N evertheless, the current results have poten­tia lJy important implications for our understanding o f desmosome co mposition and assembly during epithelial morphogenesis . It is clear that at least one DSG and one DS C are required to form d esmosomes in epithelial tissues, but our data sugges t that the p airing of individual DSG and DSC subtypes may not be as restri c ted as was previously thought. In those strati fie d epithelial ce lJs e xpressing DSGl without DS C 1, the type 1 D sg may combine w ith other D scs such as D sc2 or D sc3, which are kno wn to be expr essed be fore D sc1 in embryonic epithelia (I A King, unpub­lish e d observations). Thus, epithe lial diffe rentiation and keratiniza­tion may be associated with complex overlapping expression patte rns of individu al DSG and DSC sub types, giving rise to a co ntinuum of desmosomal cadherin combinations with g raduall y chan ging adhesive properties. The current data suggest that D sg1 may b e more " promiscuo us" in its pairing th an Dsc1 , implying important fun ctional diffe ren ces between the two desmosom al cadhe rin classes, possibly re lated to th e longer cyto plasmi c domains in th e D sgs. Recent transfectio n ex periments in A43 1 cells using chlmaeric constructs of desmosomal cadherin cyto plasmic tail s fused to the tran smembrane porti on of connexin 32 have also sugges ted diffe rent roles for these do mains in D sgs and Dscs . T he Dsgl chimera produced a domjnant-negative effec t causing disas-

DSC I EX PRESS ION IN D EVELO PM ENT 537

Skin Tongue

Figure 8. ImU1unofluorescent staining of embryonic mouse skin and tongue with the Dsc l specific antibody B13. Frozen sections o f E15.5 (n) , E16.5 (/J). and E17.5 m o use skin (c) o r E17 .5 (d) . E19.5 (f) . and n e wbo rn 111 0 U SC ton g ue OJ 'were incuba te d w'i th B 13 h ybr idol11<l supe rna ta nt containing 0 .:1 o/., e th ylencdiamine tc traacctic acid and bound anti bodies were detected with flu orescein iso thiocya nate- conjuga ted rabbi t 3nti­mouse IgG. Senle bnr, 100 J.Llll .

sembly of endogeno us desm osomes and de tachment o f inte rmedi­ate filaments. In contrast, D sc1 a chimeras fo rmed gap-like junc­tions and induced the assembly of struc tures resembl ing the desmosome plaq ue that contain ed plakoglobin and desm oplakin (T royan ovsky el a/ , 1993) . T he reasons fo r these diffe ren t effects are not ye t clear, bu t they do poin t to q ui te specific ro les fo r the cytoplasmic domains of D sg1 and Dsc1 in their dyn amic in terac­tions with various components of the intracellular pl aqu e and / or the intermediate filam ent ne twork of the cytoskeleton .

lIVe thnllil ItVfll" )' Hnll"" jor prfpnrill.~ rill' ell/br)'o secriolls jor ill situ h),I)I;<ii::nri()I(.

R.EFER E N CES

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